Methods of stimulating immune response in virally infected individuals

ABSTRACT

The present invention is directed to a method of increasing the time period between outbreaks of genital herpes comprising providing an imidazoquinolinamine formulation, disposing an amount of the imidazoquinolinamine formulation into a first nare of an individual infected with Herpes Simplex Virus type 2, covering at least a portion of the internal surface of the individual&#39;s first nare with a portion of the amount of the imidazoquinolinamine in the nare, massaging the portion of the amount of the imidazoquinolinamine into the internal surface of the first nare, disposing the amount of the imidazoquinolinamine formulation into a second nare of the individual, covering at least a portion of the internal surface of the second nare with a portion of the amount of the imidazoquinolinamine in said nare and massaging the portion of the amount of the imidazoquinolinamine into the internal surface of said nare.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 12/113,833 which is a continuation-in-part of U.S.application Ser. No. 11/318,659, filed Dec. 21, 2005, which is acontinuation-in-part of U.S. application Ser. No. 11/109,553, filed Apr.19, 2005, which is a continuation-in-part of U.S. application Ser. No.10/751,371, filed Jan. 5, 2004, which claims priority from U.S.Provisional Application No. 60/438,431, filed Jan. 6, 2003, all whichare incorporated by reference in their entirety. This application isalso being filed simultaneously with U.S. patent application Ser. No.13/031,581, entitled “Method of Treating Genital Herpes”, which isincorporated herein by reference.

TECHNICAL FIELD

The present invention generally relates to methods of stimulating animmune response in certain individuals. Methods to stimulate a hostimmune system against viral infections associated with common colds aredisclosed. Methods to stimulate immune response of a virally infectedindividual through an immunomodifier such as a non-nucleosideimidazoquinolinamine (heterocyclic amine) are disclosed. Methods tostimulate immune response of an individual infected with influenza arealso disclosed. Methods to stimulate immune response of individualshaving various cancers, such as lung cancer, bronchogenic carcinoma,bronchoalveolar carcinoma and nasal type natural killer T cell lymphomaare disclosed. Methods to stimulate immune response of individualshaving herpes simplex virus type 2 (“HSV-2”), the most common cause ofrecurrent genital herpes are disclosed.

BACKGROUND OF THE INVENTION

Uncomplicated cases of viral infections usually produce mild symptomssuch as nasal discharge, obstruction of nasal breathing, swelling of thesinus membranes, sneezing, sore throat, cough, and headache. Thesesymptoms generally last between one and two weeks. A mild infection isgenerally associated with the rhinoviruses and the coronaviruses. Theuncomplicated infection is most often referred to as the “common cold.”

At present, only symptomatic treatment is available for uncomplicatedviral infections, “common colds.” The treatments include the use ofover-the-counter decongestants, cough suppressants, cough expectorants,aspirin, and acetaminophen. The treatments, however, do not cure or evenshorten the duration of the illness. Moreover, many of the treatmentshave side effects such as drowsiness, dizziness, insomnia, or upsetstomach. Because of the diversity of the viruses, vaccines may not beeffective in preventing the onset of colds.

It has been estimated that in the course of a year, individuals in theUnited States suffer one billion colds. Colds thus have a tremendoussocietal cost in lost work days and lost school days. People suffersymptomatic discomfort. Even people receiving symptomatic treatmentstill suffer from some discomfort and additionally suffer side effectsof treatment. Moreover, influenza affects many people, typically causingmore serious symptoms than a cold.

Aldara™ (imiquimod; manufactured by 3M Corporation, St. Paul, Minn.)cream, is a prescribed patient-applied topical cream for treatingexternal genital and perianal warts. The Aldara™ product label does notrecommend using it for any other purposes.

Genital herpes is a significant health problem worldwide and continuesto increase in prevalence in the United States. In individuals in theUnited States over 12 years old, HSV-2 seroprevalence has increased from16.4% in 1976 to 21.8% in 1994 and is still rising. Thus, the currentincidence of genital herpes caused by HSV-2 in the United States isroughly one in four or five adults, with approximately 50 million peopleinfected with genital herpes and an estimated 0.5 million new genitalherpes infections occurring each year.

HSV-2 causes periods of active disease—presenting as painful blisterscontaining infectious virus particles—that last 2-21 days and arefollowed by remission when the sores disappear. Most cases of genitalherpes are asymptomatic, although viral shedding may still occur. HSV-2is transmitted by direct contact with a sore or body fluid of aninfected individual. After initial infection, the virus moves to sensorynerves, where it resides as a life-long, latent virus. The virus liesdormant in lumbrosacral that supply sensation to the genitals, perineumand upper legs.

Occasionally, these viruses reactivate and return to the area of skininfected during the primary infection. Intermittent reactivation ofviral replication can result in both symptomatic and subclinicalrecurrences because HSV-2 can reside latently in the sensory ganglion.The reactivation rate is highly variable and very difficult to predictfrom individual to individual. Many believe cell-mediated immunity playsa major role in restricting reactivation yet this role is not currentlywell understood.

Treatments are available to reduce the symptoms and speed up the healingprocess of herpes infections but there is currently no cure. Antiviraldrugs, such as aciclovir and valaciclovir, taken orally, reduce viralreproduction and shedding, and some topical creams, such as Docosanoland Tromantadine prevent the virus from entering the skin. Some otherdrugs reduce herpetic symptoms by synergising with oral antiviralmedication. Cimetidine and probenecid can reduce aciclovir clearance andaspirin can reduce inflammation associated with viral infection.

While the above listed treatment methods are effective, they requireconstant daily dosing of medications which can be expensive.Additionally, once discontinued, the medications have no post-treatmenteffect and the patient immediately reverts back to pre-treatmentreactivation levels. Therefore, it would be advantageous to develop atreatment for HSV-2 that would be effective in treating viral outbreakswhen they occur and would reduce the rate of outbreak recurrences afterthe treatment is discontinued.

In 2002, Timothy W. Schacker et al. hypothesized that long term HSV-2treatment could be achieved through enhancing the host's ability tocontrol the virus and that topical application of imiquimod in the formof Aldara™ to herpes lesions could achieve this result. Timothy W.Schacker et al., Imiquimod 5-Percent Cream Does Not Alter the NaturalHistory of Recurrent Herpes Genitalis: a Phase II, Randomized,Double-Blind, Placebo-Controlled Study, Antimicrobial Agents andChemotherapy, October 2002, pp. 3243-48. Schacker et al.'s hypothesiswas based on the knowledge that imiquimod produces a localizedimmunological response in the area of skin to which it is applied asexplained above and successful results in studies where imiquimod wasapplied to herpes lesions in Guinea Pigs. To test their hypothesis,Schacker et al. performed a phase II, randomized, double-blind,placebo-controlled study on 235 human patients. The results of the studyshowed that there was no statistically significant difference inoutbreak recurrence times between those patients treated with imiquimodand those treated with placebo. Therefore, Schacker et al. concludedthat imiquimod was not effective in reducing the rate of herpesoutbreaks in humans.

SUMMARY OF THE INVENTION

Methods to reduce the duration of symptoms associated with the commoncold, viral rhinitis or influenza, without producing any substantialside effects generally associated with symptomatic treatment aredisclosed. To reduce the duration of symptoms associated with the commoncold or influenza, methods relate to applying an imidazoquinolinamineformulation, such as, for example, an imiquimod salve within a person'snostrils, also referred to as nares. Any suitable imidazoquinolinamineformulation can be used to reduce the duration of symptoms associatedwith the common cold, viral rhinitis or influenza. Further, in anotherembodiment, the method can be used to treat various cancers includinglung cancer, bronchogenic carcinoma, bronchoalveolar carcinoma and nasaltype natural killer T cell lymphoma.

Application of imiquimod to the inside of the nostrils and in particularto the mucosal membrane of an infected individual stimulates host cellsto secrete chemical substances such as interleukins and interferons thatpromote the individual's immune response.

A method to reduce the duration of symptoms associated with the commoncold or viral rhinitis includes application of ½ packet of Aldara™(imiquimod formulation; 0.25 g of 5% active ingredient) into bothnostrils (nares) every 12 hours for a total of 4 applications. Theformulation may be applied by way of an applicator or any other suitablemeans. The formulation is applied into both nares at the onset of thecold. The onset is the day when the first cold symptoms appear. If theformulation is not applied on the first day the symptoms appear, itshould be applied by the next day. The formulation is applied twicedaily for two consecutive days. The formulation can be massaged into theinternal surface of each naris. The treatment of the second naris isafter the treatment of the person's first naris.

An imiquimod formulation is applied as described above at the onset offirst cold symptoms such as nasal irritation, watery eyes, nasal drip orother early cold symptoms. The earlier the imiquimod formulation isapplied after the onset of the cold, the shorter the recovery from thecold. An imiquimod formulation may also be applied the next day afterthe onset of the cold.

A method to reduce the duration of symptoms associated with the commoncold or viral rhinitis includes application of a coating of the mucosalmembrane within each naris with Neosynepherine® (phenylephrine) prior toapplying the Aldara™ formulation within each naris. The Neosynepherine®(phenylephrine) may be applied in the form of an over-the-counter liquidformulation by means of a spray bottle. The Neosynepherine®(phenylephrine) is preferably applied 15 minutes before applying theimiquimod formulation.

Another aspect of the present invention is directed to a method ofincreasing the time period between outbreaks of genital herpescomprising providing an imidazoquinolinamine formulation, disposing anamount of the imidazoquinolinamine formulation into a first nare of anindividual infected with Herpes Simplex Virus type 2, covering at leasta portion of the internal surface of the individual's first nare with aportion of the amount of the imidazoquinolinamine in the nare, massagingthe portion of the amount of the imidazoquinolinamine into the internalsurface of the first nare, disposing the amount of theimidazoquinolinamine formulation into a second nare of the individual,covering at least a portion of the internal surface of the second narewith a portion of the amount of the imidazoquinolinamine in said nareand massaging the portion of the amount of the imidazoquinolinamine intothe internal surface of said nare. The imidazoquinolinamine formulationis first applied within 12 hours after an appearance of first symptomsand is reapplied every 12 hours thereafter until it has been appliedfour times to each nare.

Other novel features, characteristics and aspects of the methodsdescribed herein can be further understood with reference to the belowdescribed drawings, detailed description, examples, and the appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are provided to illustrate some of the embodiments of thedisclosure. It is envisioned that alternate configurations of theembodiments of the present disclosure maybe adopted without deviatingfrom the disclosure as illustrated in these drawings.

FIG. 1 pictorially illustrates how cytokines promote and regulate theimmune cell response;

FIG. 2 pictorially illustrates further how cytokines help to regulateand promote the body's immune response;

FIG. 3 a shows a side and top perspective view of a swab-type applicatorfor use with an imiquimod formulation

FIG. 3 b shows a side and top perspective view of a spray nozzle coupledto a bottle; the spray nozzle is the applicator for a liquid imiquimodformulation.

FIG. 3 c shows a cross-sectional side view of an injection tubeinterfaced with a hollow swab head which can be used to apply animiquimod formulation; the injection tube is connected to a vessel andthe vessel has a piston actuator to inject a certain amount of imiquimodthrough the swab head into a naris.

FIG. 3 d shows a side and perspective view of a dropper-type nozzlewhich dispenses liquid imiquimod in droplet form; the nozzle isconnected to a squeeze bulb.

FIG. 4 pictorially illustrates a possible mode of action of animidazoquinolinamine such as imiquimod in stimulating host immunesystem.

FIG. 5 shows the structural formulae for imiquimod and resiquimod.

FIGS. 6A-6B show a time course of IFN-a (A) and TNF-a (B) inductionfollowing nasal application of imiquimod (n=5) □ or only base cream incontrols (n=3) □.

FIG. 7. shows the kinetics of cytokine induction by nasal application ofimiquimod in macaques (n=4). ▴ IFN-a;  TNF-a.

FIG. 8. shows intranasal expression of IFN-a mRNA by nasal applicationof imiquimod. Boxes show all results in a group. Medians indicated byhorizontal bars; S.D. by vertical bars. Level of significance is givenfor mRNAs in relation to albumin mRNAs by using quantitative RT-PCRassays (p=0.004).

DETAILED DESCRIPTION

While the concepts of the present disclosure are illustrated anddescribed in detail in the drawings and the description below, such anillustration and description is to be considered as exemplary and notrestrictive in character, it being understood that only the illustrativeembodiment is shown and described and that all changes and modificationsthat come within the spirit of the disclosure are desired to beprotected.

FIG. 1 generally describes how some cells in the human body operate aspart of the host immune system to combat infection. In FIG. 1, alymphocyte (monocytic dendritic cell) 10 takes in an antigen 11 anddisplays part of the digested antigen 13 with a marker molecule 12 to amature T cell 13. The T cell secretes cytokines 14 which help stimulatethe B cell to mature into a plasma cell 15 which produces antibodies 16.The foreign antigen in the present diagram is viral. This is known asT-helper 2 mode.

This figure is a schematic representation of the adapted immune systemwhich works much more slowly than the innate immune system. As part ofthe innate immune system, the skin and mucous membranes have been shownto be able to produce and secrete cytokines such as TNF-a, and the like.FIG. 2 discloses a macrophage 20 digesting a foreign antigen 21. Themacrophage 20 displays antigen fragments 21 a on its marker 22 to animmature T cell 23. Cytokines 24 are produced and help the T cellmature. Further cytokines 24 actually produced by the maturing T cellhelp the maturing T cell evolve into killer cells 25 and helper T cells26. Cytokines 24 also help attract additional macrophages 27,granulocytes 28, and other lymphocytes to the area of infection therebypromoting an attack on infected cells 29 (this is now known as T-helper1 mode).

Imiquimod enhances both the innate and adapted cell-mediated immunepathways to stimulate the production of various cytokines. For example,imiquimod stimulates the innate immune response by inducing thesynthesis and release of cytokines, including IFN-a and TNF-a in bothhumans and animal studies. Inhalation of imiquimod results in evenlarger amounts of IFN-a and TNF-a, to the point of creating flu-likesymptoms. Production of various cytokines by the activated innate immunesystem results in the strengthening of the cell-cell interaction. Forexample, monocytes, macrophages, B cells, and dendritic cells (includingLangerhan cells; LC) are targeted by imiquimod.

A proposed mechanism by which imiquimod may activate the above-mentionedtarget cells is via the activation of Toll-like receptors (TLRs), afamily of pathogen recognition receptors located on the cell surface ofvarious innate immune cells such as dendritic cells. Activation of TLRs,such as, for example, TLR7 results in the downstream activation of asignal cascade mediated by MyD88 and various effector cytokines such asIFN-a, IL-12, and IL-18 are produced (FIG. 4).

A proposed mechanism of action for imiquimod to activate thecell-mediated immune response is through an indirect stimulation of Tcells by producing Th-1 cytokine IFN-γ. Imiquimod also enhances themigration of LCs to the regional lymph nodes to enhance antigenpresentation to T cells. In vitro assays have established that exposureof LCs to imiquimod results in increased gene expression for TF-a,IL-1∃, and IL-12, and also secretion of IFN-(by imiquimod-treated Tcells compared to untreated cells.

Studies have shown that immune response modifiers such as imiquimod andresiquimod are TLR7 agonists and induce type 1 interferon in numerousspecies including humans. Imiquimod and resiquimod induce IFN-a frompurified plasmacytoid dendritic cells.

Thus imiquimod and resiquimod stimulate the local production of variouscytokines such as IL-12, IL-18, IL-1∃, IFN-a, and IFN-( to promote bothinnate as well as cell-mediated immunity.

The common cold causes a group of symptoms that usually are easilyrecognized by patients and doctors. About 50 percent of patients willdevelop a sore throat, which is often the first symptom to appear sinceit can occur as early as 10 hours after infection. This is followedrapidly by the most common symptoms of the common cold—congested nasaland sinus passages, a runny nose and sneezing. Hoarseness and cough areless likely to occur, but they may last longer than other symptoms,sometimes for several weeks.

Most patients diagnose the common cold by the typical symptoms of runnynose, congestion and sneezing, and rarely consult medical attention.Symptoms typically peak on the second, third or fourth days of infectionand last about one week to 10 days. Up to 25 percent of people may havepersistent symptoms, such as a nagging cough that can last for severalweeks.

The methods disclosed herein stimulate the immune system response asdescribed in FIGS. 1, 2, and 4. The methods disclosed herein promotehost cells to secrete chemicals and cytokines such as interferons andinterleukins, which impact the host cellular immune response at leastpartially as shown in FIGS. 1, 2, and 4.

Low molecular weight heterocyclic non-nucleoside imidazoquinolinaminescan be used to treat viral rhinitis. One such imidazoquinolinamine isimiquimod whose IUPAC nomenclature is(1-(2-methylpropyl)-1H-imidazo)[4,5-c]quinolin-2-amine. Imiquimod mayalso be referred to as R-837. Another imidazoquinolinamine isresiquimod, whose IUPAC nomenclature is4-amino-2-ethoxymethyl-a,a-dimethyl-1H-imidazol[4,5-c]quinoline-lethanol. Resiquimod may also be referred to as R-848.(see FIG. 5).

To provide effective treatment, a formulation of imiquimod commonly usedto treat warts can be used. For example, the formulation sold in salveformat under the brand name Aldara™ is effective. It is believed thatother imiquimod formulations such as imiquimod in a fluid formulation orin a fine powder formulation might be effective.

A method to reduce the duration of symptoms associated with the commoncold or viral rhinitis includes application of ½ packet of Aldara™(imiquimod formulation; 0.25 g of 5% active ingredient) into bothnostrils (nares) every 12 hours for a total of 4 applications. Each gramof 5% Aldara™ cream contains 50 mg of imiquimod as active ingredient.

In an embodiment, an applicator is used to disperse the imiquimod withineach naris. The applicator used can be a cotton swab. See FIG. 3 a. Theswab should be of suitable size to fit internally within each naris suchthat the exterior of the swab can move freely within each naris and makesubstantial contact with the naris' mucosal membrane. In an embodiment,the imiquimod salve is combined with the swab shown in FIG. 3 a byapplying a 4 mm³ dab of the imiquimod salve on the head of the swab. Theswab is then inserted in a naris and moved around within the naris so asto spread the salve over the naris' mucosal membrane.

In another embodiment, the foregoing methods utilizing imiquimod mayalso be used to treat influenza. The applicator is used to disperse theimiquimod within each naris. The applicator used can be a cotton swab.See FIG. 3 a. The swab should be of suitable size to fit internallywithin each naris such that the exterior of the swab can move freelywithin each naris and make substantial contact with the naris' mucosalmembrane. In an embodiment, the imiquimod salve is combined with theswab shown in FIG. 3 a by applying a 4 mm³ dab of the imiquimod salve onthe head of the swab. The swab is then inserted in a naris and movedaround within the naris so as to spread the salve over the naris'mucosal membrane. For treating influenza, the imiquimod may be appliedonce a day for two days. The nares are then squeezed against the septumto improve absorption.

In a further embodiment, the foregoing methods utilizing imiquimod mayalso be used to treat various cancers such as lung cancer, bronchogeniccarcinoma, bronchoalveolar carcinoma and nasal type natural killer Tcell lymphoma. The applicator is used to disperse the imiquimod withineach naris. The applicator used can be a cotton swab. See FIG. 3 a. Theswab should be of suitable size to fit internally within each naris suchthat the exterior of the swab can move freely within each naris and makesubstantial contact with the naris' mucosal membrane. In an embodiment,the imiquimod salve is combined with the swab shown in FIG. 3 a byapplying a 4 mm³ dab of the imiquimod salve on the head of the swab. Theswab is then inserted in a naris and moved around within the naris so asto spread the salve over the naris' mucosal membrane. The nares are thensqueezed against the septum to improve absorption.

The swab shown in FIG. 3 a is not the only type of applicator which canbe used to apply an amount of imiquimod salve to a naris. Many othertypes of applicators can be used. For instance, an applicator with ahollow swab head fluidly connected with an injection tube would work.See FIG. 3 c. The hollow swab head 30 would have a series of tinyapertures 31 through which the salve could be extruded. Extrusionthrough the tiny holes would occur by way of actuating an amount ofsalve in the injection tube 33 to flow into the hollow swab head ′30.Actuation could occur by a plunger 34. Once the salve is extruded, thehollow swab head is moved around within the naris so as to spread thesalve within the naris. The swab head could have many configurations.Additionally, it is feasible that one could use an injection tube aloneto dispense the salve in the naris. The salve could then be spread by acotton swab, a finger or some other means.

Instead of using imiquimod in a salve formulation, one may also useimiquimod in a liquid formulation. If an imiquimod liquid formulation isused, the applicator can be a spray nozzle 40 a, FIG. 3 b, or a droppernozzle 40 b, FIG. 3 d. The nozzles 40 a, 40 b could be interfaced tovessels such as bottles 41 a or squeeze bulbs 41 b. The vessels 41 a, 41b and nozzles 40 a and 40 b would be configured so that a predeterminedactuation sequence would emit an effective dosage of imiquimod from thenozzle into the naris. For instance, to emit imiquimod from the spraynozzle 40 a, an operator would simply depress pump 42 interfaced withthe spray nozzle. In addition to the above, the applicator could simplybe a finger or any other member which would fit within a naris and allowdispersion of the imiquimod formulation within the naris.

It is preferable after first applying the imiquimod formulation to theinternal surface of a naris or nares, i.e., within the naris, to massagethe formulation into the naris' mucosal membrane.

Each of a person's two nares is treated in the same fashion. Treatmentof the second naris is immediately after the first naris. Massaging ofthe salve into at least a portion of the internal surface of each nariscan occur after the salve has been applied to both nares.

Prior to treating each naris with the imiquimod formulation, each nariscan be precoated with (a solution of about 10% phenylephrinehydrochloride) at least 15 minutes before applying the imiquimodformulation. Prepping the nares with the formulation facilitatesprolonged contact of the imiquimod to the nares' internal surfaces byhelping to prevent wash-off due to nasal secretion. Phenylephrine is adecongestant that works by constricting (shrinking) blood vessels (veinsand arteries). Constriction of blood vessels in the sinuses, nose, andchest allows drainage of these areas, which decreases congestion. Anyother suitable alpha-adrenergics or other decongestants may also beused.

The utility of the above-described method for treating persons withviral infections can also be seen by reference to the below in vivoexperiments.

In each of the tests, an imiquimod salve was used. The formulation wasthat commonly used to treat warts and sold under the brand name Aldara™.The salve was applied to each naris of the test subject by use of acommon cotton swab. Either a 4 mm3 dab of salve or ½ pack of 5% Aldara™was placed on the swab head. The swab head was inserted into a naris.The swab was moved around inside the naris to distribute the salve overthe mucosal membrane of the naris. Immediately, after application of theAldara™ to the subject's first naris, a swab was used to apply theAldara™ to the subject's second naris. Immediately after application toeach naris, the salve was massaged into the mucosal membrane of eachnaris.

Application of an immunomodifier such as imiquimod to the internalsurface of the nostrils stimulates innate immunity locally and thushelps to shorten the duration of cold symptoms.

EXAMPLES Example 1 Alleviation of Viral Rhinitis Symptoms byAdministering Imiquimod at or about the Onset of the Cold Symptoms

A test sample of six patients was treated for viral rhinitis usingimiquimod (5% topical cream Aldara™, manufactured by 3M Corporation, St.Paul, Minn.). The patients were diagnosed with viral rhinitis due toinitial symptoms such as congested nasal passages (rhinitis), nasal dripor rhinorrhea, and sneezing. Contents from ½ packet of a standardimiquimod formulation such as, for example, Aldara™ were applied with acotton swab into both nares by massaging gently but thoroughly.Approximately the contents from ½ packet of 5% Aldara™ was appliedthoroughly along the inside surface of both nares. Initial applicationof Aldara™ to all the six patients occurred within 24 hours after theappearance of first symptoms resembling viral rhinitis, in order tomaximize the efficiency of imiquimod in stimulating the immune systemwhen the viral load is presumably smaller. The procedure was repeatedevery 12 hours for up to 48 hours. The imiquimod packets wererefrigerated after opening, and the remaining contents were used forsubsequent applications.

The patients were monitored for changes in the viral rhinitis symptoms.No untoward side effects were reported by any of the patients throughoutthe course of the treatment. The first sign of relief (reduced nasalcongestion, nasal drip) was obtained between 12 and 36 hours afterbeginning the imiquimod treatment. Complete disappearance of symptoms(nasal congestion, sore throat, headache; malaise) was obtained within48 hours of treatment. One patient with ulcerative colitis (a possibleTh2-type disorder) did not suffer any further aggravation during thetreatment. In an unrelated incident, during the course of treatment, onepatient inhaled imiquimod and developed severe flu-like symptoms thatspontaneously subsided within 24 hours. Therefore, care should be takennot to inhale the imiquimod formulation during application in thenostrils.

The results demonstrate that an imiquimod formulation is effective inreducing the duration of symptoms during viral rhinitis or common cold(TABLE 1). The imiquimod and other related compounds such as resiquimodstimulate the immune cells both locally and also systemically to mount adefense response against the viruses. The cold symptoms subsided within48 hours compared to about a week or 10 days for untreated viralrhinitis. Formulations of imidazoquinolinamines such as, for example,imiquimod or resiquimod can thus be effectively used to mitigatesymptoms during viral rhinitis. Depending on the intensity of the viralrhinitis, an imiquimod or resiquimod formulation or the treatment plancan be modified. For example, instead of every 12 hours, the imiquimodformulation can be applied every 8 hours. In addition, appropriatemodifications of the amount of imiquimod can also be undertaken.Furthermore, any suitable method of administration can be implemented,such as, for example, using a swab, or a drip applicator or as a nasalspray.

Example 2 Alleviation of Viral Rhinitis Symptoms by AdministeringImiquimod after the Onset of the Cold Symptoms

Infected Test Subject #1 developed a sore throat with tingling inlarynx, pharynx and uvula. 12 hours later, Subject 1 also developedcongestion of the nose. One day later, the subject's initial symptomsintensified, and the subject further developed systemic symptoms such asmalaise and headache. More than twenty four hours after the initialviral rhinitis symptoms appeared, Aldara™ was applied in each nostrilwith a Q-tip swab.

A 4 mm³ dab of salve was placed on the swab head. The swab head wasinserted into a naris. The swab was moved around in the naris so as todistribute the salve over the mucosal membrane of the naris.Immediately, after application of the Aldara™ to the subject's firstnaris, a swab was used to apply the Aldara™ to the subject's secondnaris. Immediately after application to each naris, the salve wasmassaged into the mucosal membrane of each naris. The subject was alsotreated with 2 teaspoons of standard cough suppressantguaifenesin/dextromethorphan (Wal-Tussin). The next day, malaise andheadache were more pronounced and Aldara™ was reapplied with Q-tip toeach nostril as described above. Also, 2 teaspoons ofguaifenesin/dextromethorphan was administered.

Two days after the first application of Aldara™, marked improvement ofsymptoms, including malaise and headache was observed. Three days afterthe first application of Aldara™, some cough and rhinorrhea persisted,and by 4-5 days, all of the cold symptoms subsided.

Infected Test Subject #2 developed common cold with rhinorrhea and nosecongestion. Aldara™ was applied once to each nostril every day for 3days. By day three, except for post nasal drip, other cold symptomssubsided.

It was noted the effectiveness of the treatment decreased markedly ifthe imiquimod formulation was applied more than 2 days after the onsetof the cold symptoms. Compared to Example 1, the Aldara™ treatmentdescribed in Example 2 required longer duration for complete symptomrelief (see TABLES 1 and 2). This may be due to factors such as (i)delayed application of Aldara™ after the onset of the first coldsymptoms, and (ii) infrequent application (once a day compared to twicea day in Example 1). Therefore, immediate application of Aldara™ or anyother imiquimod formulation after the onset of the cold symptoms mayresult in quicker relief of cold symptoms.

It was also noted by the inventor and the inventor's wife, empiricallyon themselves, that if treatment occurred within 12 hours of onset ofsymptoms, the infection was terminated overnight. It is believedtreatment may need to occur within six hours of onset to terminate theinfection. It was also noted that the application of Aldara™ for morethan two days may result in severe irritation of the nasal mucosa,possibly mediated by TNF-a.

TABLE 1 Viral rhinitis and treatment data with imiquimod PatientSymptoms Day 0^(a) Day 1 Day 2 Patients 1-6 Local Symptoms: PresentSubstantial Complete sore throat; reduction reduction nasal congestion;rhinitis; rhinorrhea Systemic Symptoms: Present Substantial Completeheadache; reduction reduction cough; malaise ^(a)onset of first coldsymptoms and first administration of Aldara ™. Thereafter, Aldara ™ wasadministered every 12 hours for 48 hours.

TABLE 2 Viral rhinitis and treatment data with imiquimod delayedadministration of imiquimod after the first onset of cold symptoms.Patient Symptoms Day 0^(a) Day 1^(b) Day 2 Day 3 Day 4 Test LocalPresent Present Substantial Only mild Complete Subject 1^(c) Symptoms:reduction rhinorrhea reduction sore throat; nasal congestion; rhinitis;rhinorrhea Systemic Present Present Slight Substantial CompleteSymptoms: reduction reduction reduction headache; cough; malaise TestLocal Present Substantial Only mild Complete Subject 2^(d) Symptoms:reduction rhinorrhea reduction sore throat; nasal congestion; rhinitis;rhinorrhea Systemic Present Slight Substantial Complete Symptoms:reduction reduction reduction headache; cough; malaise ^(a)onset offirst cold symptoms. ^(b)first administration of Aldara ™. Thereafter,Aldara ™ was administered every 24 hours up to 3 days. ^(c)coughsuppressant was also administered. ^(d)Aldara ™ was administered on Day0, when the cold symptoms first appeared. Thereafter, Aldara ™ wasadministered every 24 hours up to 3 days.

Example 3 Prolonged Remission Period Between Outbreaks of Genital Herpesby Administration of Imiquimod During Outbreaks

A test sample of four patients was treated for HSV-2 using imiquimod (5%topical cream Aldara™, manufactured by 3M Corporation, St. Paul, Minn.).The patients were diagnosed with HSV-2 due to typical outbreak symptomssuch as lesions. For each patient, contents from ½ packet of a standardimiquimod formulation such as, for example, Aldara™ were applied with acotton swab into both nares by massaging gently but thoroughly. Thus,the contents were applied thoroughly along the inside surface of bothnares. Initial application of Aldara™ to all the four patients occurredwithin 12 hours after the appearance of first symptoms resembling anoutbreak of genital herpes. The application procedure was repeated every12 hours up to 48 hours so that a total of four applications wasadministered to each patient. The imiquimod packets were refrigeratedafter opening, and the remaining contents were used for subsequentapplications.

Prior to treatment with Aldara™, the four patients were having herpesoutbreaks at fairly regular intervals. Patients 1, 2 and 3 wereexperiencing outbreaks every 2 to 3 months and Patient 4 was havingoutbreaks once a month. Each of the four patients then began treatmentby undergoing the 48 hour Aldara™ regimen detailed above each time he orshe experienced an outbreak. After undergoing 2 to 4 regimens, eachpatient experienced a prolonged period of remission before the nextoutbreak. Patient 1 did not have another outbreak for approximately 3.5years, Patient 2 did not have another outbreak for approximately 6months, Patient 3 did not have another outbreak for approximately 10months and Patient 4 did not have another outbreak for approximately 2months. See Table 3 below summarizing these results.

TABLE 3 Genital Herpes treatment data with imiquimod Pre-TreatmentRemission Post-Treatment Remission Patient Interval (Approximate)Interval (Approximate) Patient 1 2-3 months 3.5 years Patient 2 2-3months   6 months Patient 3 2-3 months  10 months Patient 4   1 month  2 months

The results demonstrate that an imiquimod formulation is effective inreducing the frequency of genital herpes outbreaks and increasing thelength of the intervening remission period between outbreaks. Theseresults are unexpected and surprising in light of the teachings andconclusions of Schacker et al. who found imiquimod was ineffective inprolonging HSV-2 remission in humans. The discrepancy may be explainedby noting that imiquimod and other related compounds such as resiquimodwhen used in conjunction with the present invention stimulate a systemicimmunological response to mount a defense response against HSV-2.Imiquimod's ability to produce a localized immunological response hasbeen long known and led to Schacker et al.'s hypothesis that applying itto herpes lesions would increase remission intervals. However, althoughunexpected, it appears HSV-2 can be controlled by generating a systemicimmunological response as provided by the present invention.

The invention is further described with reference to the following assaydemonstrating the effectiveness of imiquimod on Macaque monkeys. In theassay, messenger RNA levels of interferon (IFN)-a were quantified.Quantification demonstrated 2-5 fold increases in nasal secretionsfollowing a single nasal application of imiquimod as compared withuntreated macaques. The assay also indicated a rapid induction of IFN-a1-5 hours post treatment, and a proportional increase of tumor necrosisfactor (TNF)-a which remained 3 times above the controls even at 6 hpost nasal treatment. No adverse reactions to treatment were found inmacaques when the cream was used during this short period of time. Theassay demonstrates that nasal application of imiquimod rapidly induceshigh levels of IFN-a and TNF-a production and therefore may limit theacquisition of the virus.

The assay was limited to examining nasal secretions following nasalapplication (both nares) of imiquimod, because it is believed that thisis the entry site where an immune response will be first observed. Theassay evaluated the appearance of mRNA IFN-a and TNF-a in Macaca mulattamacaques and compared it with a control group treated with a base cream(without imiquimod) or simply anesthetized for the same time interval asthe treated group. ELISA measurements of IFN-a and TNF-a (FIGS. 6A-6B)showed a very significant increase (p<0.0001) over time of bothcytokines (IFN-a to 630 ng/mg protein (FIG. 6A); TNF-a to 540 ng/mgprotein (FIG. 6B) as compared with a minimal and steady concentration ofIFN-a and TNF-a in the control group (FIGS. 6A-6B).

Kinetic studies of IFN-a and TNF-a levels showed a very rapid inductionof IFN-a even at 1 h post treatment increasing to 5 times basal level at3 h, remaining near maximal level at 4 h, but decreasing rapidly to nearbasal level at 6 h post treatment. TNF-a also increased proportionally,but slightly less than IFN-a peaking at 4 h, but remaining high even at6 h post treatment. The significance of the data is two-fold. First,intranasal administration of imiquimod cream (5%) produced enough IFN-ain a short period of time, so the cream can be applied daily in order tohave 5-6 h of good efficacy. Thus, for example, one can begin applyingthe cream prior to encountering a known viral area such as an airplane.However, the cream has known cytotoxic effects (mediated through thecytokine production), especially if applied for weeks, so extendedapplication would not be recommended. In the assay, TNF-a remained highfor at least 6 h. In the treated group of macaques, the cream was washedaway with warm water after the experiments. No animal had cytotoxiceffects when examined at 6 h, 12 h, or 24 h, except one animal, whichhad an episode of lacrimation for 12 h post treatment. The cream needsto be applied to induce maximum concentration of IFN-a, but it may beremoved when TNF-a continues to be elevated (after 6 h).

The assay also employed a second set of tests to evaluate mRNA IFN-ainduction by imiquimod with postnasal swabs. mRNA was isolated andanalyzed by quantitative competitive RT-PCR. An internal standardconstructed to be complementary to and to compete with oligonucleotideprimers and for amplification of target sequences was used. Samplestaken 3-5 h after postnasal application were used. IFN-a proteinexpression was induced by imiquimod at levels 2-5 times the controlsamples. (FIG. 7) mRNA IFN-a was also variably induced in control animalsamples (anesthesia only) at ratios between 0.01-0.55 with a median of0.192. The treated animals expressed IFN-a protein at a level of0.84-5.20 mRNA×10³ with a mean of 2.3 IFN-a/albumin mRNA×10³ (FIG. 3).The difference was significant (p=0.004) irrespective of whether IFN-amRNA levels were related to albumin, 13 actin or GAPDH as referencetranscripts.

To perform the assay, two groups of Indian Macaca mulatta verified to befree of simian immunodeficiency virus (SW) and simian retrovirus type D(SRV) infections were used. The control group (n=3) and the treatedgroup (n=5) were sedated with glycopyrolate 0.01 mg/kg+acetaminophen 0.2mg/kg and anesthetized with zolazepam (Telazol) 10 mg/kg intramuscular.The animals were then shaved under the nares and sample “0” from thepostnasal fluid was taken by inserting thin sticks with cotton swabsdeep into the nares. The swabs were then re-inserted in the sterilecollection tubes containing transport media Hank's balanced saltsolution with 10% glycerol and 200 U/mg each of penicillin andstreptomycin, 250 mg/ml gentamycin and 50 U/mg nystatin. In thetreatment group, ½ packet of Aldara™ cream was massaged gently in eachnaris. A packet of Aldara™ contains 0.25 g imiquimod and a base creamconsisting of isostearic acid, cetyl alcohol, white petroleum,polyphorbate 60, glycerin, benzyl alcohol, and propylparaben. In thecontrol group, only the cream was massaged. Samples were taken atdifferent times and stored at 4° C. for 1 day and at −20° C. for oneweek. Physical examination after each sample and at 6 h, 12 h, and 24 hpost anesthesia consisted of examining each macaque for fever, erythema,erosion, flaking, and lacrimation. The animals were followed for 1 weekfor change in weight, eating habits, stool consistency, and fever.

RNA was isolated with a high pure RNA isolation kit (Roche, MolecularBiochemicals) according to the instructions from the manufacturer.

Quantification by Competitive RT-PCR. Two mg total cellular RNA wasreverse transcribed. Quantification of cDNA corresponding to transcriptsof interest was performed by using internal cDNA standards (IS). Inbrief, IS were constructed to be complementary to and compete witholigonucleotide primers and for amplification of target sequences.Target cDNA were amplified in the presence of 10- and two-fold serialdilutions of the IS. The amount of target transcripts was thencalculated on the basis of the known molecular quantity of the IS, andrelated to the amount of a reference mRNA (albumin, [3-actin, orglycerinaldehydephosphate dehydrogenase (GAPDH)), which had beenquantified in parallel.^(18,19)

IFN-a Primer Sequences

SEQ ID NO: 1 5′-GAAGCTTYCTCCTGYYTGAWGGACAGA-3′ SEQ ID NO: 25′-GGG GA TCCTCTGACAACCTCCCANGCACA-3′

(annealing temperature 68; # cycles 36; mRNA size product 372 bp; ISsize 506 bp).

Albumin Primers

SEQ ID NO: 3 5′-CTTGAATGTGCTGATGACAGG-3′ SEQ ID NO: 45′-GCAAGTGAGCAGGCATCTCATC-3′

IFN-a and TNF-a enzyme-linked immunoabsorbent assays (ELISA). Levels ofIFN-a and TNF-a were measured using commercially available multispecieskit (PBL Biomedical Laboratories) (product #41105-1). The range ofdetection was 10-500 pg/ml (high sensitivity protocol).

Statistical analysis. Data were compared using student t-test or one-wayANOVA and Dunnett's comparison test. Differences were consideredsignificant at p<0.05.

A More powerful Imidazoquinolinamine is in the pipeline (e.g.Reziquimod). I expect it to be even more powerful as an antiviral agentfor the same purposes.

1. A method of stimulating an immune response in an individual affectedwith influenza, the method comprising essentially of: providing animidazoquinolinamine formulation; disposing an amount of theimidazoquinolinamine formulation into a first naris of a virallyinfected individual; and covering at least a portion of an internalsurface of the individual's first naris with a portion of the amount ofimidazoquinolinamine in said first naris; wherein theimidazoquinolinamine formulation is applied within 12 hours after anappearance of first symptoms.
 2. The method of claim 1 furthercomprising: disposing a second amount of the imidazoquinolinamineformulation into a second naris of the virally infected individual; andcovering at least a portion of an internal surface of the individual'ssecond naris with a second portion of the second amount ofimidazoquinolinamine in said second naris.
 3. The method of claim 1,wherein the imidazoquinolinamine formulation's active ingredient isselected from the group consisting of imiquimod and resiquimod.
 4. Themethod of claim 1, wherein the imidazoquinolinamine formulation isdisposed using a device selected from the group consisting of a swab,syringe, spray nozzle, and a drip applicator.
 5. The method of claim 1,wherein the imidazoquinolinamine formulation comprises imiquimod as anactive ingredient in the amount of about 3 mg.
 6. The method of claim 1,wherein the imidazoquinolinamine formulation comprises resiquimod as anactive ingredient in the amount equivalent to about 3 mg of imiquimod 7.The method of claim 1, wherein the imidazoquinolinamine formulation isselected from a group consisting of a cream, gel, liquid, paste,aerosol, and an emulsion.
 8. The method of claim 1, wherein about 15minutes prior to disposing the imidazoquinolinamine formulation, thefirst nare is treated with a decongestant.
 9. The method of claim 7,wherein the decongestant is Neosynepherine.
 10. The method of claim 1further comprising providing the imidazoquinolinamine formulation everytwelve hours until relief from cold symptoms is obtained.
 11. A productfor stimulating an immune response in an individual affected with theflu consisting essentially of: an imidazoquinolinamine formulation; andan instruction set, wherein the instruction set directs the individualto dispose an amount of the imidazoquinolinamine formulation into afirst naris of the individual and to cover at least a portion of theinternal surface of the first naris with a portion of theimidazoquinolinamine in the naris.